Evaluation of Ia expression in rat ocular tissues following inoculation with interferon-gamma

It is becoming increasingly clear that IFN-gamma is a potent immunoregulatory protein which influences MHC class II (Ia) antigen expression and cellular functions of B cells, T cells, NK cells and macrophages. During the past 5 yr our laboratory has provided evidence that IFN-gamma modulates class II antigens on retinal cells (retinal pigment epithelial cells, endothelial cells) and is localized within the eye during human inflammatory conditions. In this study we evaluate the direct effect of IFN-gamma on ocular tissue. Lewis rats were inoculated intravitreally or under the retina with either recombinant IFN-gamma (20,000 U) or saline. At 2 hr, 1, 2 and 6 days postinoculation, the eyes were removed and frozen sections were evaluated by immunocytochemical staining with monoclonal anti-Ia antibodies and an irrelevant monoclonal anti-T cell antibody. Saline treated tissue and tissue removed 2 hr after IFN-gamma inoculation showed no significant staining for Ia antigens. However, eyes evaluated 24 hr after IFN-gamma inoculation revealed Ia expression on a variety of ocular cells localized in the conjunctiva and anterior segment, such as conjunctival epithelium, keratocytes, iris epithelium, ciliary epithelium and choroidal cells. In the retina, retinal pigment epithelial (RPE) cells were Ia positive only when IFN-gamma was injected directly under the retina. In conjunction with Ia expression, two striking changes were noted. An iritis was seen and infiltrating cells were detected in the inner retinal layers. Both of these phenomena have been observed in certain inflammatory eye diseases. These studies clearly substantiate the concept that IFN-gamma can regulate class II antigens in the eye and thus may perpetuate immune reactivity in this site.     

Anti-Ia antibody diminishes ocular inflammation in experimental autoimmune uveitis

An experimental model of inflammatory eye disease, experimental autoimmune uveitis (EAU), was established by injecting rats in the footpad with S-antigen in complete Freund's adjuvant. This model system was used to evaluate the role of major histocompatibility complex (MHC) class II antigens (Ia) in the pathogenesis of this T cell mediated disease. One day prior to S-antigen priming, rats were injected with either anti-Ia antibodies or with mouse ascites. Clinical and histopathological analysis of eyes from rats treated with anti-Ia antibody showed less ocular inflammation as well as a delay in onset of EAU when compared to controls (p = 0.01). Furthermore, immunocytochemical evaluation demonstrated that tissue obtained from animals receiving anti-Ia therapy also expressed less Ia antigen, as well as a diminution in the number of infiltrating macrophages and lymphocytes. These data show that anti-Ia treatment significantly modifies the course of EAU in the rat. In addition, this study suggests that MHC class II antigen expression may be involved in the initiation and continuation of immune responses that results in ocular inflammatory diseases.     

Expression of HLA-DR antigen on retinal pigment epithelial cells in retinitis pigmentosa

Class II (HLA-DR) antigens are cell surface molecules that play a major role in the initiation and perpetuation of immune responses. Although most cells do not constitutively express class II antigens, selected cells can be stimulated to do so in some immunologically mediated disorders. When retinal pigment epithelial cells were evaluated by either immunoperoxidase or immunofluorescent staining of frozen eye sections from normal individuals, HLA-DR antigens were not detected. In contrast, retinal pigment epithelial cells from two patients with retinitis pigmentosa did express HLA-DR antigens. These findings demonstrated that at some time during the course of retinitis pigmentosa, the retinal pigment epithelial cell is activated to express HLA-DR.     

Cellular immune response to Ia induction by intraocular gamma-interferon

BALB/c mice and Lewis rats were injected intraocularly with recombinant gamma-interferon (IFN-gamma). The distribution of Ia antigens and cellular infiltrates was studied over a 21-day time course. In mice, Ia antigens were induced on cells in the corneal stroma, corneal endothelium, iris, ciliary body, retinal pigment epithelium and cells in the retina. No detectable cellular infiltrate accompanied class II major histocompatibility (MHC) antigen induction in mice. In rats, cells in all of the intraocular tissues mentioned above were induced to express Ia antigens. However, in distinct contrast to mice, the induction of Ia antigens on intraocular structures in rats was accompanied by intense cellular inflammation composed predominantly of polymorphonuclear cells. These findings demonstrate that aberrant Ia expression on intraocular tissues by IFN-gamma is not sufficient to elicit a cellular autoimmune response in mice. The observed species-related differences in cellular immune response to class II MHC antigen induction on intraocular tissues parallels the susceptibility of mice and rats to experimental autoimmune uveitis.     

Antigen presentation of herpes simplex virus by corneal epithelium--an in vitro and in vivo study.

Ia antigen (class II antigen) is a histocompatibility antigen that foreign peptides associate with, before antigen presentation to T cells and subsequent triggering of the CD4 T cells. Although corneal epithelium is normally Ia negative it may become Ia positive under abnormal circumstances but the functional significance of this is uncertain. In this study the expression of Ia antigen on corneal epithelium of mice during in vivo primary and secondary herpes simplex keratitis and the in vitro accessory function of corneal epithelium in the presentation of herpes simplex virus (HSV) antigen to in vivo HSV primed T cells were evaluated. Whole mount preparations of corneal epithelium were found to express Ia antigen on days 3, 5, and 7 following corneal inoculation with live HSV. The intensity of the Ia expression was greater in non-immune mice on day 7 after corneal inoculation compared with immune mice. A cellular suspension of corneal epithelium induced HSV primed T cells to proliferate in the presence of HSV antigen. Induction of Ia antigen on corneal epithelium during herpes simplex keratitis may functionally expand the population of antigen presenting cells in the cornea and contribute to T cell activation.     

Modulation of HLA antigen expression on conjunctival fibroblasts by gamma-interferon

We investigated the ability of recombinant human gamma-interferon (rhIFN-gamma) to induce class II HLA antigen expression on human conjunctival fibroblasts in cell culture. Cultures were established by explanting subconjunctival tissue from normal donor globes. Fibroblasts were treated with rhIFN-gamma at concentrations ranging from 1 to 500 units/ml and incubated for 1, 3 and 6 days. HLA antigens were detected by immunofluorescence using monoclonal antibodies in conjunction with flow cytometry. Class I antigen was identified using a monoclonal antibody directed against Beta-2 microglobulin (a component of the class I antigen complex). Class II histocompatibility antigens were detected using monoclonal antibodies specific for HLA-DR, HLA-DP and HLA-DQ. Class I antigen was present on all cells prior to induction and showed a trend toward increased density after treatment with rhIFN-gamma. Class II antigens were absent before induction with rhIFN-gamma. After treatment with rhIFN-gamma, class II antigens were induced in a dose- and time-dependent fashion. HLA-DR expression was most sensitive to induction by rhIFN-gamma, followed by HLA-DP, and then HLA-DQ. The up-regulation of HLA class I antigen expression and the inducible expression of class II antigens following exposure to rhIFN-gamma suggest that conjunctival fibroblasts have the potential to participate in immunologic diseases of the external eye.     

Expression of HLA class I and II antigens on cells of the human trabecular meshwork

Human corneoscleral tissue containing trabecular meshwork and cultured human trabecular cells were examined for HLA-ABC (class I) and HLA-DR (class II) antigens of the major histocompatibility complex using an indirect immunofluorescence assay. Class I antigens were detected in the trabecular meshwork on frozen sections and on cultured trabecular cells. Class II antigens were constitutively expressed on some, but not all, cells within the trabecular meshwork. Many more cells could be induced to express class II antigens by pre-incubation in human gamma interferon. Cultured trabecular cells did not express class II antigens constitutively, but expression could be induced by gamma interferon. This study suggests that, in addition to Langerhans' cells at the limbus, other cell types within the anterior segment express major histocompatibility complex-encoded class II antigens either constitutively or inducibly. These cells may be important for the initiation and regulation of ocular immunity.     

HLA antigenicity of normal and pathological corneas

Fresh human corneas and corneal buttons were studied for expression of HLA antigens. Using monoclonal antibodies in an indirect immunofluorescence assay, corneal layers were examined for class I (HLA-A, B, C) and class II (HLA-DR) histocompatibility antigens. Twenty-one human corneas were studied, 6 normal and 15 pathological: 4 buttons of allograft rejection, 9 buttons of pseudophakic bullous keratopathy. In fresh control corneas, HLA-A, B, C antigens were localized on corneal epithelium and on stromal keratocytes but were never found on endothelial cells. HLA-DR antigens were not detected on corneal epithelium, stroma or endothelium but were detected on Langerhans cells within epithelium and anterior stroma. At the corneal limbus, HLA class I-II antigens were expressed on vascular endothelium. HLA antigen distribution was modified in pathological corneas. Antigens HLA-A, B, C were induced on endothelial cells of rejected corneal allografts. Antigens HLA-DR were detected on epithelial cells, cells in the stroma of pseudophakic bullous keratopathy and also on endothelial cells of rejected corneal allografts. These results suggest that induction of class I and II antigen expression by inflammatory factors may occur in vivo. In rejected corneal allografts induction of HLA-DR antigen on corneal layers would intensify the process of rejection. This study and others have demonstrated the ability of modulation of HLA antigen expression on human corneal cells in vivo.     

Class II antigens on retinal vascular endothelium, pericytes, macrophages, and lymphocytes of the rat

Class II histocompatibility complex antigens on the retinal vascular endothelium may allow these cells to function as antigen-presenting cells to circulating T cells. The present study investigated induction of class II antigens in vitro to characterize the response under controlled conditions. Retinal vascular endothelium from Lewis and Brown Norway rats (high versus low responders in experimental autoimmune uveitis) were exposed in vitro to recombinant rat gamma interferon, interleukin-1, interleukin-2, or Concanavalin-A spleen supernatant. Retinal pericytes, macrophages and lymphocytes were studied in comparison. A newly adapted ELISA technique was used to assay levels of antigen expression. Class II antigens (I-A OX6, I-E OX17, polymorphic I-A OX3), class I antigens (OX18), macrophage marker (OX42), macrophage and T helper cell marker (W3/25), and T suppressor/cytotoxic cell marker (OX8) were studied. Results showed that retinal vascular endothelium normally expresses very little class II antigen. However, high levels of I-A and I-E were induced by interferon or spleen supernatant. The levels of class II antigen approached that of the traditional antigen-presenting cell (macrophage) and were much higher than levels for pericytes and lymphocytes. The same doses of interferon showed larger increases in the Lewis rat compared to Brown Norway. No effect was seen with interleukin-1 or -2. Therefore, retinal vascular endothelium may be induced by gamma interferon to express class II antigens with degree of induction greater than or equal to the macrophage, and higher levels of induction were seen in the high responder strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferative vitreoretinopathy membranes. An immunohistochemical study

Proliferative vitreoretinopathy (PVR) is the leading cause of failure after retinal detachment surgery. Therefore, both the extracellular matrix and cellular components of preretinal membranes from 23 eyes with PVR were characterized immunohistochemically. The membrane stroma was composed primarily of types I, II, and III collagen. Laminin and both heparan sulfate proteoglycans and collagens types IV and V were co-distributed in discrete regions within the stroma. Glial and retinal pigment epithelial (RPE) cell populations were identified in these membranes using specific immunohistochemical markers as was a small but significant macrophage population. Double-labeling experiments indicated that RPE cells in these membranes expressed the class II histocompatibility antigen HLA-DR, although neither the RPE monolayer in situ nor cultured RPE cells was HLA-DR positive unless induced by gamma interferon. Only rare isolated vascular endothelial cells were detected in 5 of the 23 membranes.     

Expression of Ia antigen by ocular tissues of mice treated with interferon gamma

Intraperitoneal injections of interferon gamma (IFN-gamma) induced or enhanced the expression of Ia antigen by selected murine ocular tissues. In contrast, topical application of this lymphokine had no effect. Ia antigen expression was noted in most cellular components of the conjunctiva, iris, ciliary body, choroid and sclera of treated mice. Intense staining was also found in stromal cells of the cornea, but no Ia reactivity was detected in the corneal epithelium. The lack of staining in the corneal epithelium is in contrast to the strong reactivity in the adjacent conjunctival epithelium, as well as in the skin epithelium. There was no increase in the number of Langerhans cells in the peripheral cornea and limbus, or in their intensity of staining with the anti Ia antibodies. The pattern of Ia expression in ocular tissues of mice treated with IFN-gamma is similar to that of rats with experimental autoimmune uveoretinitis (EAU) except that Ia is expressed more regularly and with higher intensity by the retinal vascular endothelium of rats with EAU than by that of IFN-gamma-treated mice. The findings in the current study thus support the notion that IFN-gamma may be involved in immune-mediated inflammation in the eye.     

Interferon-gamma induces the expression of major histocompatibility antigens by human retinal glial cells.

Retinal glial cells normally do not express major histocompatibility (MHC) molecules. However, the expression of MHC antigens by retinal glial cells under certain pathological conditions suggests that these cells may play a role in immune responses of the retina. At present, the identity of molecules that may regulate the expression of class I and class II MHC antigens by retinal glial cells has not been established. Since interferon-gamma (IFN-gamma) induces MHC antigens in a variety of cell types, we examined the effect of this lymphocyte product on class I and class II antigen expression by human retinal glial cells in culture. Glial cells derived from post-mortem donor eyes were exposed to recombinant human IFN-gamma. Cells with MHC class I or class II antigens were detected with the use of specific monoclonal antibodies that were labeled directly or indirectly with fluorescent probes. Fluorescence of labeled cells was assayed by flow cytometry. Exposure to IFN-gamma increased the expression of class I and class II antigens by the retinal glial cells. Double labeling studies showed that 90% of the glial cells induced to express class II antigens also had class I antigens. Overall, the results show that IFN-gamma can regulate the expression of MHC molecules by human retina glial cells in culture. These findings are consistent with the concept that glia may play a role in immunological disorders affecting the retina.     

Cryopexy enhances experimental autoimmune uveoretinitis (EAU) in rats

Treatment of rat eyes with cryopexy enhanced the development of experimental autoimmune uveitis (EAU) in these eyes. The enhancement of EAU by cryopexy was particularly pronounced when the disease was induced by active immunization in rats of a low responder strain (Wistar Furth), or by adoptive transfer with lymphocytes sensitized against S-antigen. The disease enhancement was expressed by earlier onset of clinical symptoms and by more severe inflammatory changes. Histological examination of cryopexy-treated eyes showed focal necrosis and inflammation, confined to the affected sites. Immunohistochemical analysis of the inflammatory infiltration revealed it consists mainly of macrophages and T-lymphocytes of the helper and suppressor subsets. In addition, increased expression of class II antigens was observed in affected areas, on both inflammatory and resident ocular cells. Using electron microscopy and Evans blue angiography we could show breakdown of the blood-retinal barrier at the treated sites. Histological examination of eyes with EAU following cryopexy showed localization of the early inflammation at the injured site. The data are interpreted to suggest that the enhanced EAU in cryopexy-treated eyes is mainly due to the breakdown of the blood-retinal barrier, the accumulation of lymphoid cells and the increased expression of class II antigens, which facilitates antigen presentation.     

Cross-matches on donor cadaver retinal pigment epithelial cells in corneal risk patients

Background Allografts can be rejected either through the antibody-mediated or cellular pathways. The objective of this study was to look at the extent of antibody formation in patients awaiting re-keratoplasty using cross-matches on cadaver retinal pigment epithelial (RPE) cells. Methods CadaverRPE cells were derived by trypsin digestion from donor eyes (n=1200). After 3 days of cell cultivation, the cells were adherent and began to lose their pigment. By day 7 most cells were clear and grew as a polygonal monolayer. MHC class I expression by RPE cells was studied by the W6/32 (anti-HLA-A, B, C) monoclonal antibody (MoAb) and that of class II (HLA-DR) by the 136 MoAb. Normal RPE cells express few class I and no detectable class II antigens. For the induction of MHC expression, cells were subsequently stimulated with 250 U/ml of recombinant gamma interferon for 5 days. Cells were used for tissue typing and also for cross-matches with recipient serum. Cross-matches were subsequently performed and measured by flow cytometry. Results Both class I and class II antigens were strongly enhanced, as could be shown by immunohistochemical staining. Some 20% of those patients awaiting rekeratoplasty (n=60) were positive for anti-HLA antibodies. In one case anti-DR3 antibodies were detected in a recipient who had had several rejection episodes after keratoplasty. Conclusions RPE cells are not only useful for cadaver post-mortem HLA typing but also for donor-specific cross-matches. The degree of antibody formation after keratoplasty in rejecting patients was, however, low. This may imply that anti-HLA antibodies are not the major cause of corneal graft loss after keratoplasty.     

Detection of HLA class I and II antigens in rejected human corneal allografts

We compared the distribution of HLA-ABC (class I) and HLA-DR (class II) antigens on fresh human donor corneal tissue, donor corneas following a 72-hour storage in McCarey-Kaufman (M-K) medium, and corneal buttons from patients with allograft rejection and with chronic herpetic stromal keratitis. Incubation in M-K media had little or no effect on the distribution of HLA antigens as compared with fresh tissue. In contrast to control corneas, both HLA class I and II antigens were detected on corneal endothelial cells, cells in the stroma, and on basal epithelial cells in rejected allografts. Corneal endothelium in herpetic buttons did not express detectable HLA antigens. HLA-DR positive Langerhan's cells were demonstrated in the central corneal epithelium of rejected allografts, as well as in herpetic corneas, but not in control corneas except at the limbus. Based upon these observations, a theory of corneal allograft rejection in humans is proposed based upon the induction of class I HLA-ABC and class II HLA-DR antigens on cells in the donor button by a factor(s) associated with cellular inflammation.     

HLA-A,B,C, and HLA-DR antigens and dendritic cells in fresh and organ culture preserved corneas

Fresh and organ-culture preserved human corneas were examined for HLA-A,B,C (class I) and HLA-DR (class II) histocompatibility antigens using mouse monoclonal antibodies and an indirect immunofluorescence technique. HLA-A,B,C antigens were detected on epithelial cells and on keratocytes, but not on endothelial cells in fresh corneas. The expression of HLA-A,B,C antigens was not significantly altered by organ-culture for a period up to 7 days. The epithelial cell layer bearing the larger part of the HLA-A,B,C antigens decreased, however, from 6-7 layers to 2-3 during organ culture. HLA-DR antigens were not detected on any of the corneal layers, but were present on scattered dendritic cells within the corneal epithelium and on cells in the corneal stroma just beneath the Bowman's membrane. Using immunofluorescence, no cells bearing HLA-DR antigens were seen in cornea sections obtained after 1 week in organ-culture. These results demonstrate that the presence of HLA antigens, particularly of the HLA-DR antigens, is affected by the organ-culture preservation of the human cornea.     

Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.     

Expression of class II antigen in endotoxin induced uveitis

Uveitis can be induced by systemic or intravitreal administration of endotoxin (lipopolysaccharide, LPS). In this study we correlated the expression of class II antigens (la in rat) of the Major Histocompatibility Complex (MHC), with this experimental model of uveitis. Ia antigen was detected by immunohistochemistry using the Avidin-Biotin-Peroxidase Complex (ABC) method and the monoclonal antibody OX6. Ia antigen was not expressed in normal eyes. However, Ia was expressed in the anterior uvea epithelial cells in all eyes with LPS induced uveitis. This study demonstrates that the ocular Ia expression is a localized process in the anterior uvea in response to systemic or intravitreal LPS. This response appears to be distinct from the action of LPS on macrophage Ia expression, where LPS has been shown to inhibit the induction of Ia antigen in macrophages by gamma interferon.     

Experimental autoimmune uveitis induced by immunization with retinal pigment epithelium-specific 65-kDa protein peptides

PURPOSE: To investigate the pathogenic potential and sites of retinal pigment epithelium-specific 65-kDa protein (RPE65) for inducing experimental autoimmune uveitis (EAU) in Lewis rats. METHODS: Twenty-six peptides were chemically synthesized based on the amino acid sequences of human RPE65. These peptides spanned the entire RPE65 sequence. Each peptide was injected into a footpad and the peritoneum of Lewis rats. The eyes were examined by slit-lamp biomicroscopy, and the findings were correlated with the histological findings. The serum antibody titer and lymphocyte reactivity against each peptide was also determined by enzyme-liked immunosorbent assay (ELISA) and lymphocyte proliferation assay, respectively. RESULTS: Active immunization of rats resulted in the induction of EAU with 14 (3 severe and 11 mild) of the 26 peptides. The clinical course of the EAU was similar to that induced by the injection of retinal antigens such as S-antigen or inter-photoreceptor retinoid binding protein (IRBP). However, the histopathologic changes differed from the EAU induced by these retinal antigens. The inflammation was induced mainly from the retinal pigment epithelium (RPE) and the choroid, while the retina was relatively well-preserved except for some granulomatous changes adjacent to the RPE. CONCLUSIONS: Active immunization with peptides making up RPE65 will induce EAU. RPE65 has multiple EAU-inducing sites for Lewis rats.     

Hematopoietically derived retinal perivascular microglia initiate uveoretinitis in experimental autoimmune uveitis

Background: Hematopoietically derived cells in the retina were studied for the expression of molecules associated with antigen presentation. Methods: Bone marrow cells of (Lewis × Brown Norway) F₁ rats (LBNF₁) were transplanted to sublethally irradiated Brown Norway (BN) rats to construct chimeric rats (LBNF₁→BN). Each of 21 established chimeras received an adoptive transfer of uveitogenic Lewis T lymphocytes. Three rats were killed on each of 7 consecutive days. The right eye of each rat was processed for flat-mount preparation of the retina; the left eye of each was frozen for cryostat sectioning. All tissues were then stained with one of the following antibodies: OX-3 (Lewis-specific MHC class II marker), anti-ICAM, anti-B7-1, anti-TNF-α or anti-IL-1β. Results: Initial clinical signs of EAU appeared first on day 4; by day 6, full-blown EAU was noted. The flat-mount preparations revealed the presence of OX-3+ cells in the retina, perivascularly exhibiting dendritic morphology on day 2. These cells were observed in the retinal nerve fiber layer (NFL). No B7-1+, ICAM-1+, TNF-α+ or IL-1β+ cells were detected. Cryostat sections revealed positive cell staining of perivascular microglia and astrocytes in the retinal NFL with anti-IL-1β and anti-TNF-α antibodies. Conclusions: Since only perivascular bone marrow-derived cells are seen to express MHC class II molecules prior to onset of EAU, and since these cells also generate the cytokines IL-1β and TNF-α, it appears that initial presentation of antigen in the retina could be by these cells. Received: 21 July 1999 Revised: 30 August 1999 Accepted: 30 August 1999